Recombinant proteins are becoming increasingly important as therapeutics. Recombinant proteins can be manufactured using cell lines, for example non-human mammalian host cell lines, which are engineered to express certain human gene sequences to produce a protein of interest. After the target protein is produced in the cell culture, it can be purified through known processes to high levels of purity. However, trace amounts of contaminant proteins produced by the host cell line (called Host Cell Proteins, or HCP) can be present in the depleted cell culture.
HCP can potentially impact product quality and safety. Consequently, it is important to characterize HCP present in therapeutic products to mitigate these risks. Current characterization methods for HCP, such as two-dimensional gel electrophoresis, tend to be labor-intensive and cumbersome, which can limit the use of these methods for HCP characterization, particularly during commercial production.
Electrophoresis refers to the differential movement or migration of ions by attraction or repulsion in an electric field. Capillary isoelectric focusing (cIEF) is a high-resolution separation technique based on differences in isoelectric points (pI) of sample components and is suitable for use for separation of amphoteric substances such as amino acids, peptides and pharmaceuticals. In cIEF, proteins migrate, under the influence of an electrical field, through a pH gradient created by carrier ampholytes (CA). Ampholytes that are positively charged migrate toward the cathode, while those that are negatively charged migrate towards the anode. Consequently, the pH increases toward the cathode end of the capillary and decreases toward the anode end. When an ampholyte reaches its own pI and is no longer charged, migration ceases, resulting in the formation of a stable pH gradient. An amphoteric analyte will eventually encounter a pH at which it has a zero net charge and will therefore cease to migrate, resulting in a focusing or separation of analytes by pI. cIEF has been coupled with a number of detection techniques, including UV absorbance, laser induced fluorescence and mass spectrometry (MS).
Mass spectrometry (MS) is an analytical technique in which chemical compounds are ionized to generate charged molecules or molecule fragments which are then separated according to their mass-to-charge ratio in an analyzer using an electromagnetic field. The separated ions can then be detected and analyzed.
Atmospheric Pressure Ionization (API) sources can be used to ionize sample molecules at atmospheric pressure and then transfer the ions into a mass spectrometer. API is suitable for ionization of thermally labile samples such as polymers and peptides. Electrospray Ionization (ESI) is an API application that is frequently used for MS of thermally labile and high molecular weight compounds, such as polymers and peptides, which are low- or non-volatile.
However, on-line coupling of cIEF with ESI-MS for biological samples, especially protein digests, has been challenging. Therefore, there is a need for an improved on-line coupling of cIEF with ESI-MS for biological samples.